A Sensitive and Selective Microscale SPE method for the Determination of Prostaglandin F2a in Rat Plasma by LC/MS/MS

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Ziling Lu, Jeffrey Mazzeo, Claude Mallet, Diane Diehl [Waters]
AAPS, Salt Lake City Utah, October 26-30
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SPE Format:
Oasis MAX 5 mg µElution 96-well Plate
Prostaglandin F2a; Isoprostane; Oxidative Stress Biomarkers
Analytical Techniques:
XTerra MS C18 3.5 µm Steel 2.1 mm 30 mm
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The challenge in pharmaceutical analysis is achieving both high throughput and low detection and quantitation limits (LOD/LOQ’s). As more drug candidates show increased levels of potency, smaller dosage is required; hence the need for lower LOQ’s in order to define the pharmacokinetic profile. During pre-clinical trials of a drug candidate, sample volume is relatively low, commonly in the 50 L range. Solid phase extraction (SPE) in a 96-well plate format (1, 2) can meet the demand for low LOD/LOQ’s using less sample volume. With the assistance of a robotic liquid handler, a 96-well SPE plate can be prepared in less than a minute per sample (3). To demonstrate the utility of this low elution volume plate, we developed a method for the extraction of prostaglandin F2a from rat plasma. A 96-well low elution volume plate containing a mixed-mode anion exchange (MAX) sorbent was conditioned with methanol followed by water. Prostaglandin F2a was spiked into water or rat plasma. 8-isoprostaglandin E2 was added to the plasma as the internal standard. After the sample was loaded onto the bed, water and ammonia followed by methanol was used to wash the bed to remove interferences. The analytes were eluted from the bed with two, 25 uL volumes of acetonitrile:isopropanol with formic acid. The eluent was then diluted through the plate with a water:ammonia solution. The eluents were then analyzed by LC/MS/MS. A ten-point calibration curve was also generated with samples both in water and plasma. A generic, high throughput SPE protocol was developed on the MAX sorbent. Sample recoveries were greater than 90%. Using the low elution volume plates, there is a preconcentration factor of at least 2. The calibration range was 0.1 to 100 pg/uL and was linear for both water and plasma samples. The detection limit was 0.1 pg/uL. The novel tip design permits quantitative recovery of the extracted analytes with as little as 25 uL of elution solvent, providing as much as a 5-fold increase in concentration of the analyte from plasma. The plate allows for simple dilution of the elution solution with water prior to LC/MS/MS analysis, avoiding an evaporation and reconstitution step.

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